We plan to examine the regulation of the expression of the structural gene for PPriboseP synthetase, the rate-limiting enzyme for purine synthesis, using cultured mutant mammalian cells in continuous culture. We shall utilize cell hybridization techniques for determining dominant recessive characteristics of the regulatory mutants and for chromosome mapping of the structural gene for PPriboseP synthetase. We have developed a ultramicro peptide mapping technique to be used for delineating which domains of the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene are responsible for catalytic function and which account for the presumed regulatory role. The two-dimensional denaturing gel separation technique of O'Farrell will be used to characterize the products of the specific mutations in the cell and to isolate these molecules for peptide mapping. The hypothesis derived from our studies of mutant cells isolated in vitro will be tested by screening hyperuricemic patients for electrophoretic and isotachophoresis variants of erythrocytic PPriboseP synthetase and HGPRT activities, respectively. By generating in vitro mouse teratocarcinoma stem cell mutants with HGPRT deficiencies and injecting these mutant cells into mouse blastocysts, we anticipate the generation chimeric mice, significant components of which (including gametes) are derived from the embryonal carcinoma cells. Those chimeric males with sperm derived from the teratocarcinoma cells will be used for the siring of female carriers of the mouse Lesch-Nyhan syndrome, from which we can breed HGPRT deficient hemizygous males.